The present invention relates to methods and apparatuses for characterization of single polymers. In particular, the invention relates to methods and apparatuses for determination of the velocities of single elongated polymers. The invention also relates to methods for determination of the length and molecular mass of single polymers. The invention further relates to methods of determining the distance between landmarks on single polymers.
Analysis of the structure and dynamics of single macromolecules in a fluid sample has attracted considerable interest due in part to the rapid development of methodologies for the manipulation and detection of single macromolecules. For example, recent developments in experimental techniques and available hardware have increased dramatically the sensitivity of detection so that optical detection can be made of single dye molecules in a sample. Single dye detection can be done in aqueous solution, at room temperature (see, e.g., Weiss, 1999, Science 283: 1676-1683), and in very small volumes to reduce background. Such single-molecule based analytical methods are especially useful in the analysis of biological macromolecules, such as nucleic acid molecules and proteins. Single-molecule analytical methods require small amounts of sample, thereby alleviating tedious efforts in generating large amounts of sample material. For example, single-molecule analytical methods may allow analysis of the structure of nucleic acid molecules without amplification, e.g., by polymerase-chain reaction (PCR). Single-molecule analytical methods also allow analysis of individual molecules, and are thus particularly useful in the identification of structural and/or dynamical features without the effect of averaging over a heterogeneous population.
A single-molecule electrophoresis (SME) method which combines single molecule detection and electrophoresis has been reported for the detection and identification of single molecules in solution (Castro and Shera, 1995, Anal. Chem. 67: 3181-3186). In SME, sizing of single molecules is accomplished through determination of electrophoretic velocities by measuring the time required for individual molecules to travel a fixed distance between two laser beams. This method has been applied to DNA, to fluorescent proteins and to simple organic fluorophores. For example, SME offers a single-molecule method for sizing of DNA restriction fragments. However, SME detects only the presence or absence of a molecule. The method does not provide information regarding the internal structure of a molecule.
A single-molecule DNA sizing method using a microfabricated device has also been reported (Chou et al., 1999, Proc. Natl. Acad. Sci. USA 96:11-13). The method makes use of the fact that the amount of intercalated dye is proportional to the length of the molecule, and determines the lengths of single DNA molecules by measuring the total fluorescence intensity of DNA stained with intercalating dye molecules. Thus, the method does not use electrophoretic mobilities to determine sizes of molecules. This method also does not provide information regarding the internal structure of a molecule.
PCT Publication No. WO 98/10097 discloses a method and apparatus for detection of single molecules emitting two-color fluorescence and determination of molecular weight and concentration of the molecules. The method involves labeling of individual molecules with at least two fluorescent probes of different emission spectrum. Simultaneous detection of the two labels indicates the presence of the molecule. The velocity of the molecule is determined by measuring the time required for the molecules to travel a fixed distance between two laser beams. Comparison of the molecule""s velocity with that of standard species permits determination of the molecular weight of the molecule, which may be present in a concentration as small as one femtomolar.
Other techniques for characterizing single macromolecules include a method described in U.S. Pat. No. 5,807,677 for direct identification of a specific target nucleic acid sequence having a low copy number in a test solution. This method involves the preparation of a reference solution of a mixture of different short oligonucleotides. Each oligonucleotide includes a sequence complementary to a section of the target sequence and is labeled with one or more fluorescent dye molecules. The reference solution is incubated with the test solution under conditions favorable to hybridization of the short oligonucleotides with the nucleic acid target. The target sequence is identified in the solution by detection of the nucleic acid strands to which one or more of the labeled oligonucleotides are hybridized. To amplify the fluorescence signal, a xe2x80x9ccocktailxe2x80x9d of different oligonucleotides are used which are capable of hybridizing with sequences adjacent to but not overlapping with the target sequence. The disadvantage of this method is that, in order to design probes of the proper sequence, the exact sequence of the target nucleic acid and surrounding sequences must be known. A method described in U.S. Pat. No. 5,599,664 and European Patent No. EP 0391674 allows sizing of DNA molecules by first subjecting a DNA molecule to a force such that the DNA molecule is elongated and then measuring the conformational relaxation dynamics. In another method (Schmalzing et al., 1998, Analytical Chemistry 70:2303-2310; Schmalzing et al, 1997, Proc. Natl. Acad. Sci. USA 94:10273-10278), microfabricated devices for DNA analysis were developed, including sequencing, which employ small-scale versions of traditional techniques, such as electrophoresis.
None of these single molecule analytical methods allows the determination of the internal structure of the molecule. A challenge to the characterization of the internal structure, e.g., the linear sequence of monomers, in a single polymer chain is from the natural tendency of polymers in most media to adopt coiled conformations. The average degree of such coiling is dependent on, inter alia, the interaction of the polymer with the surrounding solution, the rigidity of the polymer, and the energy of interaction of the polymer with itself. In most cases, the coiling is quite significant. For example, a xcex-phage DNA, with a B-form contour length of about 16 xcexcm long, has a random coil diameter of approximately 1 xcexcm in water (Smith et al., 1989, Science 243:203-206).
Methods of elongating DNA molecules by fluid flow have been reported (Perkins et al. Science 276:2016-2021; Smith et al., Science 283:1724-1727). In one method, DNA molecules are stretched by an elongational flow. The probability distribution of molecular extension was determined as a function of time and strain rate. Detailed dynamics of elongated DNA molecules in elongational flow has also been observed. In another method DNA molecules are stretched by a steady shear flow. The probability distribution for the molecular extension was determined as a function of shear rate. It was found that, in contrast to the behavior in pure elongational flow, the average polymer extension in shear flow does not display a sharp coil-stretch transition.
DNA has also been stretched by electrophoresis as part of a near-field detection scheme for sequencing biomolecules. DNA has been elongated by electrophoresis both in a gel and in solution, using electrical forces to move the DNA in position for reading (U.S. Pat. No. 5,538,898). However, no data were given to determine the quality of the stretching of large polymers, and the technique is limited to analyzing approximately 3 megabases at a time.
Gravitational forces have also been used to stretch DNA (U.S. Pat. No. 5,707,797; Windle (1993) Nature Genetics 5:17-21). In this technique, drops of DNA from the sodium dodecyl sulfate lysing of cells were allowed to run down a slide held at an angle. The effect of gravity was enough to stretch out the DNA, even to its over-stretched S-DNA form. The DNA was then immobilized on the slide, making processing, e.g., fluorescent labeling, prior to stretching relatively difficult.
Single-molecule DNA analytical methods which involve elongation of DNA molecule include optical mapping (Schwartz et al., 1993, Science 262:110-113; Meng et al., 1995, Nature Genet. 9:432; Jing et al., Proc. Natl. Acad. Sci. USA 95:8046-8051) and fiber-fluorescence in situ hybridization (fiber-FISH) (13ensimon et al., Science 265:2096; Michalet et al., 1997, Science 277:1518). In optical mapping, DNA molecules are elongated in a fluid sample and fixed in the elongated conformation in a gel or on a surface. Restriction digestions are then performed on the elongated and fixed DNA molecules. Ordered restriction maps are then generated by determining the size of the restriction fragments. In fiber-FISH, DNA molecules are elongated and fixed on a surface by molecular combing. Hybridization with fluorescently labeled probe sequences allows determination of sequence landmarks on the DNA molecules. Both methods require fixation of elongated molecules so that molecular lengths and/or distances between markers can be measured.
A method for measuring the length and distances between markers on DNA was developed by Kambara et al. (U.S. Pat. No. 5,356,776). This method involves electrophoresis of DNA molecule labeled at both termini and/or internal sites through a gel, in which the DNA molecule is forced into a straight line, transferring the straightened DNA molecule into a buffer containing no gel where fluorescent labels are detected. The time interval between the detection of the two labels is used to determine the distance between them. If the two labels label the termini of the DNA molecule, the distance between the labels measures the length of the molecule. The method, which does not provide means for determining the velocity of the DNA molecule, relies on estimating the velocity of DNA from the migration rate of the DNA molecule.
Flow based single-molecule analytical methods for elongation and characterization of single macromolecules have not been widely adopted due in part to the difficulty in precise measurement of molecular characteristics, e.g., the length of the macromolecule, the distance between two landmarks on a macromolecule, etc. For example, to determine the length of an elongated macromolecule as it travels through a detection zone, e.g., a laser excitation zone, it is necessary to know the velocity of the macromolecule. The flow velocity field can be measured by various known methods, e.g., particle image velocimetry (PIV) (see, e.g., Meinhart et al., 1999, Experiments in Fluids 27: 414-419; Meinhart et al., 2000, Meas. Sci. Technol. 11:809-814). The velocities of flexible objects, such as elongated polymers, may not be the same as the flow velocities. For example, in most flows the length of a polymer may be changing as it travels along with flow. In particular, the length of a polymer may be changing as a consequence of changing flow velocity. There is therefore a need for faster, simpler, more reliable and more universally applicable methods for measuring the velocities of single elongated polymers traveling in a flow. There is also a need for more accurate methods for determining the length of single elongated polymers and/or distances between landmarks on single elongated polymers.
Citation of a reference herein shall not be construed as indicating that such reference is prior art to the present invention.
The present invention provide methods and apparatuses for determining the velocities of single elongated macromolecules. The methods of the invention are based on time-correlated measurements of an elongated macromolecule at each of a plurality of detection zones. The detection zones are located along the travel path of the elongated macromolecule at predetermined spacings. Signal amplitude profiles, e.g, intensity-time curves when fluorescence based measurements are used, of an elongated macromolecule are measured as the macromolecule passes through each of the detection zones. The measurements in the plurality of detection zones are time-correlated, e.g., synchronized, so that the temporal spacings between signal amplitude profiles measured at different detections zones are also determined.
In one embodiment, the invention provides a method for determining velocity of a single elongated polymer, said method comprising measuring a plurality of signal amplitude profiles of said elongated polymer, each signal amplitude profile comprising measurements taken at a different one of a plurality of detection zones and determining said velocity of said elongated polymer from said plurality of signal amplitude profiles, wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said elongated polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said elongated polymer at predetermined distances, and wherein said plurality of signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
In another embodiment, the invention provides a method for determining the length of a single elongated polymer, said method comprising measuring a plurality of signal amplitude profiles of said elongated polymer, each signal amplitude profile comprising measurements taken at a different one of a plurality of detection zones, and determining said length of said elongated polymer using said plurality of signal amplitude profiles and velocity of said elongated polymer, wherein each of said plurality of signal amplitude profiles comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said elongated polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said elongated polymer at predetermined distances, wherein said plurality of signal amplitude profiles are measured in a time-correlated manner, and wherein said velocity of said elongated polymer is determined from said plurality of signal amplitude profiles. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
In still another embodiment, the invention provides a method for determining the length of a single elongated polymer, said method comprising: (a) measuring a first signal amplitude profile of said single elongated polymer at a first detection zone; (b) measuring a second signal amplitude profile of said single elongated polymer at a second detection zone; (c) determining a velocity of said single elongated polymer at said first and second detection zones from said first and/or second signal amplitude profiles; and (d) determining length of said single elongated polymer by multiplying time difference between leading and trailing edges of said first or said second signal amplitude profile with said velocity; wherein each of said first and second signal amplitude profiles comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said elongated polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said elongated polymer at predetermined distances, and wherein said first and second signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the first and second signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
In still another embodiment, the invention provides a method for determining the length of a single elongated polymer, said method comprising: (a) measuring a plurality of signal amplitude profile of said single elongated polymer each at each of a plurality of detection zones; (b) determining a velocity of said single elongated polymer between each successive pair of detection zones from pair of signal amplitude profiles measured in respective pair of detection zones; and (c) determining length of said elongated polymer by (i) multiplying time interval for said elongated polymer to travel between each successive pair of detection zones with said velocity between said pair of detection zones and (ii) summing all products between time interval in one of said plurality of signal amplitude profiles; wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said elongated polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said elongated polymer at predetermined distances, said distances are shorter than length of elongated polymers, and wherein said plurality of signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
In still another embodiment, the invention also provides a method for determining a distance between a first and a second landmark on an elongated polymer, said method comprising: (a) detecting at a first detection zone said first and second landmarks on said elongated polymer; (b) detecting at a second detection zone said first and second landmark on said elongated polymer; (c) determining the velocity of said elongated polymer by dividing the distance between said first and second detection zones with time interval between detection of said first or second landmark in said first detection zone and detection of said first and second landmark in said second detection zone; and (d) determining said distance between said first and second landmark by multiplying time interval between detection of said first and second landmark at said first detection zone or said second detection zone; wherein said first and second detection zones are located in order along the path of said elongated polymer at predetermined distances, and wherein said detection in said first and said second detection zones are carried out in a time-correlated manner.
The invention also provides a method for determining velocity of a single polymer, said method comprising: (a) moving said single polymer along an elongation structure, said elongation structure comprising a tapered channel with a first end and a second end, whereby said single polymer is elongated in said tapered channel as said single polymer moves along said tapered channel from said first end to said second end; and (b) measuring a plurality of signal amplitude profiles of said single polymer, each signal amplitude profile comprising measurements taken at a different one of a plurality of detection zones and determining said velocity of said elongated polymer from said plurality of signal amplitude profiles, wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said single polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said single polymer at predetermined distances, and wherein said plurality of signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
The invention also provides a method for determining velocity of a single polymer, said method comprising: (a) moving said single polymer along an elongation structure, said elongation structure comprising a central channel holding fluid and a plurality of side channels holding fluid connected to said central channel, said central channel comprising a first end and a second end, wherein said single polymer is moved along said central channel from said first end to said second end and is elongated; and (b) measuring a plurality of signal amplitude profiles of said single polymer, each signal amplitude profile comprising measurements taken at a different one of a plurality of detection zones and determining said velocity of said elongated polymer from said plurality of signal amplitude profiles, wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said single polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said single polymer at predetermined distances, and wherein said plurality of signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
The invention also provides a method for determining velocity of a single polymer, said method comprising: (a) moving said single polymer along an elongation structure, said elongation structure comprising a channel with at least one bend, said channel comprising a first end and a second end, wherein said single polymer is moved from said first end to said second end, wherein said single polymer is moved along said channel from said first end to said second end and is elongated; and (b) measuring a plurality of signal amplitude profiles of said single polymer, each signal amplitude profile comprising measurements taken at a different one of a plurality of detection zones and determining said velocity of said elongated polymer from said plurality of signal amplitude profiles, wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said single polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said single polymer at predetermined distances, and wherein said plurality of signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
The invention also provides a method for determining velocity of a single polymer, said method comprising: (a) moving said single polymer along an elongation structure, said elongation structure comprising a channel and a plurality of obstacles to motion of said single polymer within said channel, said channel comprising a first end and a second end, wherein said single polymer moves along said channel from said first end to said second end and is elongated; and (b) measuring a plurality of signal amplitude profiles of said single polymer, each signal amplitude profile comprising measurements taken at a different one of a plurality of detection zones and determining said velocity of said elongated polymer from said plurality of signal amplitude profiles, wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said single polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said single polymer at predetermined distances, and wherein said plurality of signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
The invention also provides a method for determining velocity of a single polymer, said method comprising: (a) moving said single polymer along an elongation structure, said elongation structure comprising a first channel, said first channel comprising a first end, a second end, and a plurality of posts in a staggered arrangement between said first end and said second end, and a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end, wherein said single polymer moves along said channel from said first end to said fourth end and is elongated; and (b) measuring a plurality of signal amplitude profiles of said single polymer, each signal amplitude profile comprising measurements taken at a different one of a plurality of detection zones and determining said velocity of said elongated polymer from said plurality of signal amplitude profiles, wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said single polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said single polymer at predetermined distances, and wherein said plurality of signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
The invention also provides a method for determining length of a single polymer, said method comprising: (a) moving said single polymer along an elongation structure, said elongation structure comprising a tapered channel with a first end and a second end, whereby said single polymer is elongated in said tapered channel as said single polymer moves along said tapered channel from said first end to said second end; and (b) determining said length of said single polymer by a method comprising the steps of (i) measuring a first signal amplitude profile of said single elongated polymer at a first detection zone; (ii) measuring a second signal amplitude profile of said single elongated polymer at a second detection zone; (iii) determining a velocity of said single elongated polymer at said first and second detection zones from said first and/or second signal amplitude profiles; and (vi) determining length of said single elongated polymer by multiplying time difference between leading and trailing edges of said first or said second signal amplitude profile with said velocity; wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said elongated polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said elongated polymer at predetermined distances, and wherein said first and second signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the first and second signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
The invention also provides a method for determining length of a single polymer, said method comprising: (a) moving said single polymer along an elongation structure, said elongation structure comprising a central channel holding fluid and a plurality of side channels holding fluid connected to said central channel, said central channel comprising a first end and a second end, wherein said single polymer is moved along said central channel from said first end to said second end and is elongated; and (b) determining said length of said single polymer by a method comprising the steps of (i) measuring a first signal amplitude profile of said single elongated polymer at a first detection zone; (ii) measuring a second signal amplitude profile of said single elongated polymer at a second detection zone; (iii) determining a velocity of said single elongated polymer at said first and second detection zones from said first and/or second signal amplitude profiles; and (vi) determining length of said single elongated polymer by multiplying time difference between leading and trailing edges of said first or said second signal amplitude profile with said velocity; wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said elongated polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said elongated polymer at predetermined distances, and wherein said first and second signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the first and second signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
The invention also provides a method for determining length of a single polymer, said method comprising: (a) moving said single polymer along an elongation structure, said elongation structure comprising a channel with at least one bend, said channel comprising a first end and a second end, wherein said single polymer is moved from said first end to said second end, wherein said single polymer is moved along said channel from said first end to said second end and is elongated; and (b) determining said length of said single polymer by a method comprising the steps of (i) measuring a first signal amplitude profile of said single elongated polymer at a first detection zone; (ii) measuring a second signal amplitude profile of said single elongated polymer at a second detection zone; (iii) determining a velocity of said single elongated polymer at said first and second detection zones from said first and/or second signal amplitude profiles; and (vi) determining length of said single elongated polymer by multiplying time difference between leading and trailing edges of said first or said second signal amplitude profile with said velocity; wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said elongated polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said elongated polymer at predetermined distances, and wherein said first and second signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the first and second signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
The invention also provides a method for determining length of a single polymer, said method comprising: (a) moving said single polymer along an elongation structure, said elongation structure comprising a channel and a plurality of obstacles to motion of said single polymer within said channel, said channel comprising a first end and a second end, wherein said single polymer moves along said channel from said first end to said second end and is elongated; and (b) determining said length of said single polymer by a method comprising the steps of (i) measuring a first signal amplitude profile of said single elongated polymer at a first detection zone; (ii) measuring a second signal amplitude profile of said single elongated polymer at a second detection zone; (iii) determining a velocity of said single elongated polymer at said first and second detection zones from said first and/or second signal amplitude profiles; and (vi) determining length of said single elongated polymer by multiplying time difference between leading and trailing edges of said first or said second signal amplitude profile with said velocity; wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said elongated polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said elongated polymer at predetermined distances, and wherein said first and second signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the first and second signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
The invention also provides a method for determining length of a single polymer, said method comprising: (a) moving said single polymer along an elongation structure, said elongation structure comprising a first channel, said first channel comprising a first end, a second end, and a plurality of posts in a staggered arrangement between said first end and said second end, and a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end, wherein said single polymer moves along said channel from said first end to said fourth end and is elongated; and (b) determining said length of said single polymer by a method comprising the steps of (i) measuring a first signal amplitude profile of said single elongated polymer at a first detection zone; (ii) measuring a second signal amplitude profile of said single elongated polymer at a second detection zone; (iii) determining a velocity of said single elongated polymer at said first and second detection zones from said first and/or second signal amplitude profiles; and (vi) determining length of said single elongated polymer by multiplying time difference between leading and trailing edges of said first or said second signal amplitude profile with said velocity; wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said single elongated polymer at a plurality of times, said plurality of times comprising times that are before and after said elongated polymer is in said detection zone, wherein said plurality of detection zones are located in order along the path of said elongated polymer at predetermined distances, and wherein said first and second signal amplitude profiles are measured in a time-correlated manner. In preferred embodiments, said measuring is performed by a method comprising measuring fluorescence intensity and the first and second signal amplitude profiles are intensity-time curves. In one embodiment, the velocity determined is a center-of-mass velocity. In another embodiment, the velocity determined is a center-to-center velocity. In still another embodiment, the velocity determined is an end-to-end velocity, e.g., a leading-end-to-leading-end velocity or a trailing-end-to-trailing end velocity. In still another embodiment, the velocity determined is a rise-time velocity.
The invention further provides a method for DNA restriction fragment analysis, said method comprising: (a) moving a plurality of DNA molecules along an elongation structure, said elongation structure comprising a first channel, said first channel comprising a first end, a second end, and a plurality of posts in a staggered arrangement between said first end and said second end, and a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end, wherein said plurality of DNA molecules comprising DNA molecules generated by one or more restriction enzymes, and wherein each of said plurality of DNA molecules moves along said channel from said first end to said fourth end, separates from other DNA molecules in said plurality, and is elongated; and (b) measuring length of each of said plurality of DNA molecules by repeating for each of said plurality of DNA molecules a method comprising: (i) measuring a first signal amplitude profile of a DNA molecule in said plurality of DNA molecules at a first detection zone and a second signal amplitude profile of said DNA molecule at a second detection zone; (ii) determining a velocity of said DNA molecule at said first and second detection zones from said first and/or second signal amplitude profiles; and (iii) determining length of said DNA molecule by multiplying time difference between leading and trailing edges of said first or said second signal amplitude profile with said velocity; wherein each said signal amplitude profile comprises measurements in the respective detection zone of a signal generated at said DNA molecule at a plurality of times, said plurality of times comprising times that are before and after said DNA molecule is in said detection zone, wherein said plurality of detection zones are located in order along said second channel at predetermined distances, and wherein said first and second signal amplitude profiles are measured in a time-correlated manner.